Variables Influencing Differences in Sequence Conservation in the Fission Yeast Schizosaccharomyces pombe

Which variables determine the constraints on gene sequence evolution is one of the most central questions in molecular evolution. In the fission yeast Schizosaccharomyces pombe, an important model organism, the variables influencing the rate of sequence evolution have yet to be determined. Previous studies in other single celled organisms have generally found gene expression levels to be most significant, with numerous other variables such as gene length and functional importance identified as having a smaller impact. Using publicly available data, we used partial least squares regression, principal components regression, and partial correlations to determine the variables most strongly associated with sequence evolution constraints. We identify centrality in the protein-protein interactions network, amino acid composition, and cellular location as the most important determinants of sequence conservation. However, each factor only explains a small amount of variance, and there are numerous variables having a significant or heterogeneous influence. Our models explain more than half of the variance in dN, raising the possibility that future refined models could quantify the role of stochastics in evolutionary rate variation

Ancestral Admixture Is the Main Determinant of Global Biodiversity in Fission Yeast

Mutation and recombination are key evolutionary processes governing phenotypic variation and reproductive isolation. We here demonstrate that biodiversity within all globally known strains of Schizosaccharomyces pombe arose through admixture between two divergent ancestral lineages. Initial hybridization was inferred to have occurred ∼20-60 sexual outcrossing generations ago consistent with recent, human-induced migration at the onset of intensified transcontinental trade. Species-wide heritable phenotypic variation was explained near-exclusively by strain-specific arrangements of alternating ancestry components with evidence for transgressive segregation. Reproductive compatibility between strains was likewise predicted by the degree of shared ancestry. To assess the genetic determinants of ancestry block distribution across the genome, we characterized the type, frequency, and position of structural genomic variation using nanopore and single-molecule real-time sequencing. Despite being associated with double-strand break initiation points, over 800 segregating structural variants exerted overall little influence on the introgression landscape or on reproductive compatibility between strains. In contrast, we found strong ancestry disequilibrium consistent with negative epistatic selection shaping genomic ancestry combinations during the course of hybridization. This study provides a detailed, experimentally tractable example that genomes of natural populations are mosaics reflecting different evolutionary histories. Exploiting genome-wide heterogeneity in the history of ancestral recombination and lineage-specific mutations sheds new light on the population history of S. pombe and highlights the importance of hybridization as a creative force in generating biodiversity

Comparative structural and evolutionary analyses predict functional sites in the artemisinin resistance malaria protein K13

Numerous mutations in the Plasmodium falciparum Kelch13 (K13) protein confer resistance to artemisinin derivatives, the current front-line antimalarial drugs. K13 is an essential protein that contains BTB and Kelch-repeat propeller (KREP) domains usually found in E3 ubiquitin ligase complexes that target substrate protein(s) for ubiquitin-dependent degradation. K13 is thought to bind substrate proteins, but its functional/interaction sites and the structural alterations associated with artemisinin resistance mutations remain unknown. Here, we screened for the most evolutionarily conserved sites in the protein structure of K13 as indicators of structural and/or functional constraints. We inferred structure-dependent substitution rates at each amino acid site of the highly conserved K13 protein during the evolution of Apicomplexa parasites. We found two solvent-exposed patches of extraordinarily conserved sites likely involved in protein-protein interactions, one in BTB and the other one in KREP. The conserved patch in K13 KREP overlaps with a shallow pocket that displays a differential electrostatic surface potential, relative to neighboring sites, and that is rich in serine and arginine residues. Comparative structural and evolutionary analyses revealed that these properties were also found in the functionally-validated shallow pocket of other KREPs including that of the cancer-related KEAP1 protein. Finally, molecular dynamics simulations carried out on PfK13 R539T and C580Y artemisinin resistance mutant structures revealed some local structural destabilization of KREP but not in its shallow pocket. These findings open new avenues of research on one of the most enigmatic malaria proteins with the utmost clinical importance

Identification of individual root-knot nematodes using low coverage long-read sequencing

Root-knot nematodes (RKN; genus Meloidogyne) are polyphagous plant pathogens of great economic importance to agriculturalists globally. These species are small, diverse, and can be challenging for accurate taxonomic identification. Many of the most important crop pests confound analysis with simple genetic marker loci as they are polyploids of likely hybrid origin. Here we take a low-coverage, long-read genome sequencing approach to characterisation of individual root-knot nematodes. We demonstrate library preparation for Oxford Nanopore Technologies Flongle sequencing of low input DNA from individual juveniles and immature females, multiplexing up to twelve samples per flow cell. Taxonomic identification with Kraken 2 (a k-mer-based taxonomic assignment tool) is shown to reliably identify individual nematodes to species level, even within the very closely related Meloidogyne incognita group. Our approach forms a robust, low-cost, and scalable method for accurate RKN species diagnostics

Experimental Selection of Paromomycin Resistance in Leishmania donovani Amastigotes Induces Variable Genomic Polymorphisms

The relatively high post-treatment relapse rates of paromomycin (PMM) in visceral leishmaniasis treatment and the swift emergence of experimental drug resistance challenge its broad application and urge for rational use and monitoring of resistance. However, no causal molecular mechanisms to Leishmania PMM resistance have been identified so far. To gain insights into potential resistance mechanisms, twelve experimentally selected Leishmania donovani clonal lines and the non-cloned preselection population, with variable degrees of PMM resistance, were subjected to whole genome sequencing. To identify genomic variations potentially associated with resistance, SNPs, Indels, chromosomal somy and gene copy number variations were compared between the different parasite lines. A total of 11 short nucleotide variations and the copy number alterations in 39 genes were correlated to PMM resistance. Some of the identified genes are involved in transcription, translation and protein turn-over (transcription elongation factor-like protein, RNA-binding protein, ribosomal protein L1a, 60S ribosomal protein L6, eukaryotic translation initiation factor 4E-1, proteasome regulatory non-ATP-ase subunit 3), virulence (major surface protease gp63, protein-tyrosine phosphatase 1-like protein), mitochondrial function (ADP/ATP mitochondrial carrier-like protein), signaling (phosphatidylinositol 3-related kinase, protein kinase putative and protein-tyrosine phosphatase 1-like protein) and vesicular trafficking (ras-related protein RAB1). These results indicate that, in Leishmania, the aminoglycoside PMM affects protein translational processes and underlines the complex and probably multifactorial origin of resistance

Uncovering Natural Longevity Alleles from Intercrossed Pools of Aging Fission Yeast Cells

Quantitative traits often show large variation caused by multiple genetic factors. One such trait is the chronological lifespan of non-dividing yeast cells, serving as a model for cellular aging. Screens for genetic factors involved in ageing typically assay mutants of protein-coding genes. To identify natural genetic variants contributing to cellular aging, we exploited two strains of the fission yeast, Schizosaccharomyces pombe, that differ in chronological lifespan. We generated segregant pools from these strains and subjected them to advanced intercrossing over multiple generations to break up linkage groups. We chronologically aged the intercrossed segregant pool, followed by genome sequencing at different times to detect genetic variants that became reproducibly enriched as a function of age. A region on Chromosome II showed strong positive selection during ageing. Based on expected functions, two candidate variants from this region in the long-lived strain were most promising to be causal: small insertions and deletions in the 5'-untranslated regions of ppk31 and SPBC409.08. Ppk31 is an orthologue of Rim15, a conserved kinase controlling cell proliferation in response to nutrients, while SPBC409.08 is a predicted spermine transmembrane transporter. Both Rim15 and the spermine-precursor, spermidine, are implicated in ageing as they are involved in autophagy-dependent lifespan extension. Single and double allele replacement suggests that both variants, alone or combined, have subtle effects on cellular longevity. Furthermore, deletion mutants of both ppk31 and SPBC409.08 rescued growth defects caused by spermidine. We propose that Ppk31 and SPBC409.08 may function together to modulate lifespan, thus linking Rim15/Ppk31 with spermidine metabolism

Candidates for Balancing Selection in Leishmania donovani Complex Parasites

The Leishmania donovani species complex is the causative agent of visceral leishmaniasis, which cause 20–40,000 fatalities a year. Here, we conduct a screen for balancing selection in this species complex. We used 384 publicly available L. donovani and L. infantum genomes, and sequence 93 isolates of L. infantum from Brazil to describe the global diversity of this species complex. We identify five genetically distinct populations that are sufficiently represented by genomic data to search for signatures of selection. We find that signals of balancing selection are generally not shared between populations, consistent with transient adaptive events, rather than long-term balancing selection. We then apply multiple diversity metrics to identify candidate genes with robust signatures of balancing selection, identifying a curated set of 24 genes with robust signatures. These include zetatoxin, nodulin-like, and flagellum attachment proteins. This study highlights the extent of genetic divergence between L. donovani complex parasites and provides genes for further stud

Transient structural variations have strong effects on quantitative traits and reproductive isolation in fission yeast

Large structural variations (SVs) within genomes are more challenging to identify than smaller genetic variants but may substantially contribute to phenotypic diversity and evolution. We analyse the effects of SVs on gene expression, quantitative traits and intrinsic reproductive isolation in the yeast Schizosaccharomyces pombe. We establish a high-quality curated catalogue of SVs in the genomes of a worldwide library of S. pombe strains, including duplications, deletions, inversions and translocations. We show that copy number variants (CNVs) show a variety of genetic signals consistent with rapid turnover. These transient CNVs produce stoichiometric effects on gene expression both within and outside the duplicated regions. CNVs make substantial contributions to quantitative traits, most notably intracellular amino acid concentrations, growth under stress and sugar utilization in winemaking, whereas rearrangements are strongly associated with reproductive isolation. Collectively, these findings have broad implications for evolution and for our understanding of quantitative traits including complex human diseases

Spotsizer: High-throughput quantitative analysis of microbial growth

Microbial colony growth can serve as a useful readout in assays for studying complex genetic interactions or the effects of chemical compounds. Although computational tools for acquiring quantitative measurements of microbial colonies have been developed, their utility can be compromised by inflexible input image requirements, non-trivial installation procedures, or complicated operation. Here, we present the Spotsizer software tool for automated colony size measurements in images of robotically arrayed microbial colonies. Spotsizer features a convenient graphical user interface (GUI), has both single-image and batch-processing capabilities, and works with multiple input image formats and different colony grid types. We demonstrate how Spotsizer can be used for high-throughput quantitative analysis of fission yeast growth. The user-friendly Spotsizer tool provides rapid, accurate, and robust quantitative analyses of microbial growth in a high-throughput format. Spotsizer is freely available at https://data.csiro.au/dap/landingpage?pid=csiro:15330 under a proprietary CSIRO license